THE FOLDING AND ACTIVITY OF THE EXTRACELLULAR LIPASE OF RHIZOPUS-ORYZAE ARE MODULATED BY A PROSEQUENCE

The fungus Rhizopus oryzae synthesizes an extracellular lipase precurs or bearing N-terminal pre- and pro-sequences, Our studies in Escherich ia coli and using recombinant lipase in vitro indicate that the proseq uence of 97 amino acids has at least two functions. First, it modulate s the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form. Direct synthesis of the matu re form of the lipase in the cell has toxic consequences, at least par tly because of phospholipase activity that is suppressed in the propro tein. Secondly, it supports folding of the lipase via a pathway influe nced by a single cysteine residue at position -68. Mutational analysis of the prosequence demonstrates not only the key role of this cystein e residue but also the importance of the neighbouring amino acids, In particular, Arg-69 probably enhances the leaving group character of Cy s-68. We propose a model in which Cys-68 acts as an intramolecular thi odisulphide reagent, playing a catalytic role in the folding of the en zyme. The prosequence is capable of performing the described functions both in cis and in trans.